Dissemination of different sequence types lineages harboring blaCTX-M-15 among uropathogenic Escherichia coli in Kerman, Iran

Objective(s): Escherichia coli is one of the most important causes of urinary tract infections (UTIs). The aim of this study was to determine antimicrobial resistance, resistance and virulence genes; phylogenetic groups and identify the epidemiologic features of uropathogenic E. coli (UPEC) isolates by multilocus sequence typing (MLST). Materials and Methods: One hundred isolates of E. coli from inpatients with UTIs were collected in Kerman, Iran. Antimicrobial susceptibility testing, ESBLs, AmpC production and biofilm formation were performed by phenotypic methods. Phylogenetic groups, resistance and virulence genes were detected. Molecular typing of isolates was performed by MLST. Results: In this study, 76% of isolates were multidrug-resistant. The blaCTX-M-15 and blaTEM were the dominant ESBL-encoding gene. Among 63 ciprofloxacin-resistant isolates, the frequency of qnrS (15.8%), qnrB (9.5%), and aac (6’)-Ib (25% ) genes was shown. Fifty-five present of isolates were classified as week biofilm, (14%) moderate biofilm, and (5%) strong. The predominant phylogenetic group was B2 (3) . The prevalence of virulence genes ranged fimH (93%), iutA (66%), KpsmtII (59%), sat (39%), cnf (28%) and hlyA (27%). According to MLST results, 14 sequence types (ST) including ST-693, ST-90, ST-101, ST-1664, ST-2083, ST-131, ST-4443, ST-744, ST-361, ST-405, ST-922, ST-648, ST-5717and ST-410 were detected, indicating a high degree of genotypic diversity. Conclusion: We identified a high frequency of the ST131 clonal group among UTIs. These data show an important public health threat, and so further studies to control the dissemination and risk factors for acquisition of the ST131 clonal group and other STs are needed to make effective control.


Introduction
Escherichia coli (E. coli) is one of the most important causes of urinary tract infections (UTIs), neonatal meningitis, bacteremia, and community-and hospitalacquired infections. According to epidemiological data, almost 20% of men and 50-60% of women in their life may suffer from UTIs (1). Different antibacterial agents such as fluoroquinolones, aminoglycosides, trimethoprim/ sulfamethoxazole, and β-lactams are recommended for treatment of UTIs (2,3,4,5). The frequent use of antibiotic in treating leads to drug resistance, which results in the spread of multidrug-resistant (MDR) isolates (6). One of the most common causes of resistance to antibiotics in E. coli is the production of extended spectrum beta lactamase (ESBLs) that causes bacterial resistance to the third, and fourth-generation cephalosporins, and monobactams, especially in hospital-acquired infection. ESBL-producing uropathogenic E. coli (UPEC) are often MDR to different classes of antibiotics including aminoglycosides, and fluoroquinolones. Resistance to these agents causes delays in suitable therapy with subsequently increasing morbidity and mortality (2,6). UPEC has several virulence factors such as adhesion, fimbria (fimH), toxins (hlyA, sat, cnf), aerobactinmediated iron uptake (iutA), capsular polysaccharide (KpsmtII) and biofilm formation that lead to colonization of host mucosal surface, damage and invasion to host tissue (7,8). It also makes colonization in the bladder and causes cystitis. Studies have shown that the most important stage of infections is attachment to the host tissue (9). The ability of E. coli to adhere to uroepithelial cells is a virulence factor. Fimbria is the most important pathogenic factor in UTI; also E. coli has the ability to form biofilm in the urinary tract system, especially in the bladder (9). Recently, a relation between presence of virulence genes and E. coli phylogenetic characteristics has been reported (3). Gram negative bacteria have the ability to move through ureters to kidney and cause pyelonephritis (10). Biofilms are small colonies of bacteria surrounded by extracellular matrix that helps to the assembly and attachment of them to the tissue, causing tissue damage. More than 60% of human infections are due to biofilm. Biofilm causes bacteria resistance to the host immune system and antimicrobial agents (11).
Recent studies explained a relation between presence of virulence genes and E. coli phylogenetic characteristics. Phylogenetic groups have been determined to four major phylogenetic groups (A, B1, B2 and D) and seven subgroups A0, A1, B1, B2 (2), B2 (3), D1 and, D2 in UPEC strains (3,12,13). Extraintestinal pathogenic E. coli (ExPEC) isolates usually belong to phylogenetic groups B2, and D, and commensal E. coli isolates belong to phylogenetic groups A and B1 (13). Moreover, pathogenic extraintestinal isolates acquire specific virulence factors conferring their ability to pathogenic potential (14). Many various typing methods have been applied for identification of bacterial infection sources, as well as the prevention and control of the spread of infections (15,16). Multilocus sequence typing (MLST) is a method for molecular typing of bacteria. This method is based on 450-500 bp fragments of seven housekeeping genes loci, and the result of allelic profile is based on sequence type (ST) via a database and can be compared genetic relatedness between isolates (15,17,18). The aim of this study was to determine antimicrobial resistance profile, resistance and virulence genes, phylogenetic groups and the epidemiologic features of UPEC isolates by MLST method.

Population and bacterial isolates
Totally, 100 isolates of E. coli from inpatients with UTIs from June 2017 to June 2018 were isolated in Kerman, Iran. All isolates were identified by standard bacteriological methods (19) .
The study was approved by the Ethics Committee of Kerman University of Medical Sciences (IR.KMU.REC. 1394.327).

Detection of resistance genes by polymerase chain reaction
The boiling method was used for preparation of DNA template for polymerase chain reaction (PCR) (23) .The oligonucleotide primers were used for identifying the bla CTX-M group 1-4, bla TEM , bla SHV , bla OXA , bla PER , bla KPC and bla NDM , qnrs, qnrA, qnrB, rmtA, rmtB, rmtC, rmaA (24,25,26). PCR amplification was set up in a total volume of 25 μl containing 0.5 µl of each primer (10 pM), 12.5 µl of DNA Polymerase Master Mix RED (Ampliqon, Co, Denmark), 1 µl of DNA and 10.5 µl of water in Biometra PCR Thermal Cycler (Biometra, Germany) under the following conditions: initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 1 min, annealing at 55-64 °C for 1 min (Table  1), extension at 72 °C for 1 min and final extension at 72 °C for 5 min. In the end, PCR products were electrophoresed on 1.5% agarose gel in 0.5 TBE buffer (Tris, EDTA, Boric acid).

PCR products sequencing
The bla CTX-M positive amplification was sequenced in Macrogen, Co, South Korea. Then, the acquired nucleotide sequences were compared using online Basic Local Alignment Search Tool (BLAST) software (www.ncbi.nih.gov/BLAST program), and established as bla CTX-M-15 variant.

PCR method for the detection of virulence genes
All primers were the same as those used in previous studies (3,12,27). The PCR mixtures (25 µl) contained 1 µl of DNA, 12.5 µl of PCR master mix (Ampliqon, Inc, Co, Denmark), 0.5 µM (10 pM) of each primer and 10.5 µl of water (DNase and RNase free water). PCR amplification was comprised of the following three steps: heating at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 1 min, primer annealing at 58-63 °C for 1 min and extension at 72 °C for 1 min, followed by a final extension step of 72 °C for 5 min. Amplicons were revealed by electrophoresis on a 1.5% agarose gel, and photographed using a UV transillumination imaging system.

Biofilm assay
We analyzed the ability of the UPEC isolates to produce the biofilm according to the protocol described by Stepanović et al. (28). The Positive control for the assay was P. aeruginosa strain PAO1 and the culture medium without bacteria was used as the negative control.

Phylogenetic grouping
The distribution of phylogenetic groups in 100 UPEC isolates was determined by phylotyping PCR approach described by Clermont and colleagues (12). The results of these three amplifications allowed the classification of E. coli isolates into one of the major phylogenetic groups: A, B1, B2 or D and sub phylogenetic A0, A1, B1, B2 (2), B2 (3), D1, D2 (29).
The 50 μl of amplification reaction mixture comprised 1 µl of each primer (10 pM), 25 µl of DNA Polymerase Master Mix RED (Ampliqon, Co, Denmark), 2 µl of DNA and 21 µl of DNase and RNase free water in FlexCycler PCR Thermal Cycler (Analytik Jena, Germany). The reaction conditions were an initial denaturation step at

Statistical analysis
The SPSS software version 22.0 (IBM, Armonk, NY, USA) was used for data analysis. P-value of ≤0.05 was considered as statistically significant.

Discussion
E. coli is one of the most common bacterium in UTIs (16). Today, improper use of antibiotics has increased the resistance to various antibiotics (30). In our study, the rate of multidrug resistance among the UPEC isolates was high (76%). The highest resistance was observed against AUG, SXT, and NA (75%) and the lowest resistant was recorded against AK (6%). Also, high susceptibility was found for IMP (100%).
In recent years, MDR , AmpC and ESBL-producing E. coli isolates have been increased in different parts of the world, and it has become an emerging public health problem (31,32). In this study, prevalence of AmpC and ESBL-producing E. coli isolates have been reported 3.5%, and 55%, respectively. As previously noted, bla CTX-M genes were commonly found in large R-plasmids and carry other genes, leading to resistance to other antimicrobial agents such as fluoroquinolones and aminoglycosides (33). In our study, the rate of bla CTX-M gene and bla TEM were 74.5%. The data in this study confirms previous studies indicating that ESBL-producing E. coli isolates and β-lactamase genes such as bla CTX-M and bla TEM have been increased in different parts of the world (34,35). Four types of plasmid-mediated quinolone resistance (PMQR) (qnr, aac(6')-Ib-cr, qepA, oqxAB) have been recognized. This plasmid (PMQR) can confer resistance to multiple agents, including fluoroquinolones and ESBLs genes (36). In this study, the rate of PMQR genes was 52.3%. In our study, significant differences between the antibiotic resistant and ESBL production were detected except GM antibiotics (P<0.05). This indicates that ESBL production is important factor in antibiotic resistance. In this study, 43% of isolates were ciprofloxacin resistant and ESBL positive, and we found a statistically significant difference between resistant to ciprofloxacin and ESBL production (P=0.002). Also, we showed that fluoroquinolones -resistance genes such as qnrS (10, 23.2%), qnrB (6, 13.9%), and aac(6')-Ib-cr (25,58.1%) were strongly related with an ESBLpositivity in UPEC. This is in accordance with the results obtained by previous study (37). Biofilm production can protect bacteria from killing activity of host defense mechanisms and antibiotics. As well, it can cause expression of several virulence factors, and increase resistance against antibiotics (38). Characterization of virulence markers and drug resistance of UPEC let the physicians to follow up the development of pathogenicity of strains causing the UTIs and improving obtainable infection control policies (39). The significant differences between the biofilm formation and antibiotic resistance to CAZ (P=0.03) and AK (P=0.01) were detected. This indicates that biofilm formation can be considered as one of the factors in antibiotics resistance. Also, the prevalence of virulence genes of UPEC isolates were fimH (93%), iutA (66%), kpsMTII (59%), sat (39%), cnf (28%), and hlyA (27%). Therefore, high incidence of virulence genes could be a main causative agent for UTIs in humans. In this study, highly virulence isolates were mostly present in group B2 (3) and D (2) and less virulence strains were present in group A0. As well, we observed a statistically significant association between microbial resistance to NA (P=0.02), and CIP (P=0.008) and the phylogenic groups. So, the highest antibiotic resistance was observed in group B2 (3). In addition, statistically significant difference was observed between the presence of phylogenic groups and virulence genes of cnf (P=0.02), sat (P=0.02), and kpsMT II (P=0.001). This indicates that the highest rate of cnf, sat and kpsMT II genes virulence was observed in group B2 (3).
MLST is the best method for studying molecular epidemiology. The MLST typing determines the diversity and phylogenetic relationships of the isolates, which rely on seven housekeeping genes for each E. coli isolate that reflects population structure and evolutionary biology of bacteria. Also, this method provides comparisons between results from various laboratories (40). The portability and reproducibility of MLST will present valuable and significant information about E. coli genetic lineage in UTI (15). This study also focused on epidemiological investigation of MLST in the bla CTX-M-15 UPEC. Based on these findings, ST-131 was predominant ST-type in our hospital settings. As  complexes that are associated with UPEC  have been recognized; these are ST-693, ST-648, ST-922, ST-90, ST-361, ST-405, ST-101, ST-1664, ST-2083,  ST-5717, ST-410 and ST-744 (41). According to previous studies, discrepancy in ST131 biofilm formation is associated with bacterial culture conditions, data cut-offs and definitions used in prevalence studies and clonal diversity within each ST131 collection (42). In addition, the role of type 1 fimbriae in biofilm formation has been recognized (42). Our study supports these data. Biofilm production in ST405 was strong and prevalence of different biofilm production was reported in ST131. In the present study, ST-361, ST-744, ST-101 and ST-4433 had only one virulence factor fimH, but the rest STs had more virulence factors. In previous studies, E. coli ST131 isolates had more antibiotic resistance profile, virulence factors and biofilm production. Also, it originates from phylogenetic group B2 (3) that is related epidemiologically to extraintestinal virulence (43) . According to our present findings, one of the ST-131 was resistant to all of the antibiotics except AK, and also it had all of the virulence genes and had moderate biofilms production. This study confirmed that ST-131 has become extensively disseminated in hospital. Also, the UPEC ST-131 strain is resistant to cephalosporins, aminoglycosides and trimethoprim/sulfamehoxazole, which is considered as serious threat to public health. In this study, ST complexes that are associated with UPEC were relatively uncommon in country.
In summary, we identified a high frequency of the ST131 clonal group and prevalence of antibiotic resistance, and virulence factor among UTIs in Kerman, Iran. These factors cause competitive advantage of this clonal group, supporting its rapid worldwide dissemination. These data show an important public health threat, which necessitate further studies to control the dissemination and risk factors for acquisition of the ST131 clonal group and other STs to make effective control. Our results suggest that several ST seem to be circulating in our region.

Conclusion
As a study limitation there was only 20 isolates to detect ST. However, this study is the first report about dissemination of different STs lineage harboring bla CTX-M-15 among UPEC in Iran. We identified a high frequency of the ST131 clonal group, prevalence of antibiotic resistance, and virulence factor among UTIs. These data show an important public health threat and so further studies are needed to control the dissemination and risk factors for acquisition of the ST131 clonal group and other STs.